Production of blood coagulation factor VIII:C

ABSTRACT

A concentrate of blood coagulation Factor VIII:C is obtained in high yield by fractionation of blood plasma with a sequence of adsorption steps employing two different water-insoluble, cross-linked polyelectrolyte copolymers, each in the presence of exogenous heparin.

BACKGROUND OF THE INVENTION

This invention relates to blood fractionation and more particularly tothe production of blood coagulation Factor VIII:C.

The process of blood coagulation is a complicated physiological activitythat involves interactions of numerous substances found in normal wholeblood. It is known that certain factors associated with the bloodcoagulation mechanism are seriously deficient in certain individuals.Thus, in those patients suffering from classical hemophilia,antihemophilic factor A (AHF, Factor VIII) is deficient. In thosepatients suffering from hemophilia B, plasma thromboplastin component(PTC, Factor IX) is missing from the blood. A small percentage ofhemophiliacs also are lacking in the so-called Von Willebrand Factorwhich is an integral component of Factor VIII.

It is now generally recognized that plasma Factor VIII is a complex oftwo components that have distinct functions, biochemical andimmunological properties, and genetic control. One component of theFactor VIII complex has antihemophilic factor procoagulant activity andis usually designated Factor VIII:C. The other, larger componentcomprises the majority of the protein mass, interacts with platelets ina way that promotes primary hemostasis and is usually designated FactorVIIIR (ristocetin cofactor or Von Willebrand antigen).

Patients with Factor VIII:C deficiency transmitted by X-chromosomeinheritance (hemophilia A patients) have normal Factor VIIIR synthesisand function. Such patients thus do not require exogenous administrationof Factor VIIIR for maintenance of hemostasis, and a concentrate ofFactor VIII:C free of Factor VIIIR would be satisfactory and in somecases even preferable.

Further background information on the structure and function of theFactor VIII complex and its two components can be had by reference tothe three recent review articles by, respectively, Hoyer, J. Amer. Soc.of Hematol. 58 (1), 1-13 (1981); Harris et al, Biochim. Biophys. Acta668, 456-470 (1981); and Fulcher et al, Proc. Natl. Acad. Sci. USA 79,1648-1652 (1982).

The clinical importance of Factor VIII concentrates and the criticalneed for adequate supplies thereof has provided motivation to developimproved methods for the production of such blood fractions. Asalternatives to the conventional Cohn alcohol process of bloodfractionation which must be conducted at cold temperatures, variousother methods have been developed which employ fractionating agents thatcan be used at normal room temperature (ambient temperature). One suchmethod employs the polymer polyethylene glycol (PEG) as described, e.g.,in U.S. Pat. Nos. 3,631,018; 3,652,530; and 3,682,881. However, themethodology described in these patents additionally employs acryoprecipitation step which necessitates the use of cold temperaturefacilitates and also results in a loss of a substantial amount of theFactor VIII activity.

Addition of heparin at various stages of these PEG/cryoprecipitationfractionation processes to increase the yield of Factor VIII has beensuggested in U.S. Pat. Nos. 3,803,115; Re. 29,698; 4,203,891; and4,289,691. In the first two of these patents the heparin is added afterthe cryoprecipitation step whereas in the latter two patents it is addedbefore the cryoprecipitation step.

The aforesaid prior art methods for the production of Factor VIII by useof polyethylene glycol, cryoprecipitation and heparin are not reportedto provide a Factor VIII:C concentrate as distinguished from the FactorVIII complex. However, heparin has been suggested for addition to plasmain fractionation methodology to separate AHF, von Willebrand'sristocetin cofactor and fibronectin by cold temperature precipitationand chromatography according to U.S. Pat. Nos. 4,210,580 and 4,278,594.

Another method of improvement over the conventional Cohn alcohol bloodfractionation process employs water-insoluble, cross-linkedpolyelectrolyte copolymer adsorbents as described, e.g., in U.S. Pat.Nos. 3,554,985; 3,555,001; 4,118,554; and 4,157,431; and by A. J.Johnson, et al., J. Lab. Clin. Med. 92 (a), 194-210 (1978). Thesepolymeric materials have been employed in combination with other agentssuch as dithiothreitol, Sepharose CL-4B and Sephadex G-100 to produce aconcentrate of Factor VIII:C substantially free of Factor VIIIR. Harriset al., Biochim. Biophys. Acta 668, 456-470 (1981).

DESCRIPTION OF THE INVENTION

It has now been found that a concentrate of Factor VIII:C can befractionated from blood plasma in high yield at ambient temperature witha sequence of adsorption steps employing, at differing concentrationsand pH levels, two different water-insoluble, cross-linkedpolyelectrolyte copolymers, each in the presence of exogenous heparin.In accordance with the present invention, such concentrate of FactorVIII:C is obtained by a method which comprises:

(a) admixing blood plasma or a concentrate thereof at pH of from about7.0 to about 8.5 with from about 0.01% to about 0.1% by weight ofwater-insoluble, polyelectrolyte copolymer of ethylene and maleicanhydride cross-linked with from about 3 mole % to about 10 mole % ofloweralkyliminobis(loweralkylamine) and containing from about 90 mole %to about 100 mole % of pendant diloweralkylaminoloweralkylimidefunctional groups, said admixing in the presence of exogenous heparin,

(b) separating the supernatant from the resulting adsorbed plasmafraction,

(c) admixing said supernatant at pH of from about 5.5 to about 6.5 withfrom about 1% to about 10% by weight of water-insoluble, polyelectrolytecopolymer of ethylene and maleic anhydride cross-linked with from about3 mole % to about 10 mole % of loweralkyliminobis(loweralkylamine),containing from about 3 mole % to about 7 mole % of pendantdiloweralkylaminoloweralkylimide functional groups, and furthercharacterized in that substantially all the remaining free carboxyl oranhydride sites are blocked with alkoxyalkylamine, said admixing in thepresence of exogenous heparin,

(d) separating the resulting adsorbed plasma fraction from thesupernatant and recovering therefrom a concentrate of Factor VIII:C byelution from the adsorbent, and

(e) wherein said alkyl and alkoxy have from about one to about fourcarbon atoms.

The preferred level of heparin used herein ranges from about 0.01 toabout 2 units per ml of plasma and most preferably from about 0.1 toabout one unit per ml of plasma. The preferred concentration ofpolyelectrolyte used herein ranges from about 0.03% to about 0.04% inadsorption step (a) and from about 5% to about 6% in adsorption step(c).

As used herein, one unit of heparin is defined to mean one U.S.P.(United States Pharmacopoeia) unit. The U.S.P. unit of heparin is thatquantity which will prevent 1.0 ml of citrated sheep plasma fromclotting for one hour after the addition of 0.2 ml of a 1:100 CaCl₂solution. Heparin is generally obtained by isolation from mammaliantissues containing mast cells such as the liver and lung. As usedherein, the term "heparin" also is meant to include the pharmaceuticallyacceptable water soluble salts thereof, e.g., the sodium salt. Suitableexamples of commercially available heparin sodium products areLipo-Hepin® (Riker Laboratories), Liquaemin® Sodium (Organon), andPanheprin® (Abbott Laboratories).

The anticoagulant properties of heparin have been known since Howell'sdiscovery in 1922. Amer. J. Physol. 63, 434-435 (1922). It is now knownthat heparin acts as an anticoagulant indirectly by means of a plasmacofactor. The heparin cofactor, Antithrombin III, is an α₂ -globulin anda serine protease inhibitor that prevents the serine protease frominactivating the clotting factors. Antithrombin III forms complexes withthrombin and, as a result, both proteins are inactivated. Heparinmarkedly accelerates the velocity but not the extent of this reaction.Low concentrations of heparin increase the activity of Antithrombin IIIwhich forms the basis of heparin administration as a therapeutic.

Heparin addition to blood for collection and preservation of donor bloodis well known as seen, e.g., from Button et al., Transfusion 3, 37-40(1963). It is also known that heparin can be added to stored plasma toprevent inactivation of Factor VIII by thrombin. Rizza et al., Nature(Lond.) 180, 143 (1957) and Stibbe et al., Thromb. Diath. Haemorrh. 27,43-58 (1972). However, donor blood is now generally collected in ACD,CPD or CPD plus adenine (CPDA-1) anticoagulants instead of heparin forpurposes of erythrocyte survival. Moreover, heparinized blood isunsuitable for various tests, e.g., tests that involve complement,isoagglutinins, or erythrocyte fragility. Therefore, blood to be usedfor such tests would require removal or neutralization of heparinanticoagulants.

Although heparin has been used heretofore in connection with bloodfractionation methods which employ polyelectrolyte polymers of the typeused in the present invention, the heparin was added to the eluate fromthe adsorbent which was then fractionated with polyethylene glycol toobtain Factors II, VII, IX and X (prothrombin complex). A. J. Johnson etal., J. Lab. Clin. Med. 92 (a), 194-210 (1978). Heparin was used in theproduction of the prothrombin complex factors by polyethylene glycolprecipitation, as reported by A. J. Johnson et al., for provision of aheparin-activated Antithrombin III to inhibit activated clottingfactors. See Thromb. Diath. Haemorrh. 34, N2, 589 (1975). There has beenno suggestion in the prior art to use heparin in a blood fractionationsequence with the polyelectrolyte polymers to obtain high yields of aFactor VIII:C concentrate as defined herein.

The polyelectrolyte polymers used in combination with the heparin inaccordance with the present invention are known compounds which can bemade according to methods described in U.S. Pat. Nos. 3,554,985;3,555,001; 4,118,554; and 4,157,431. For example, the base copolymer ofethylene and maleic anhydride (EMA) can be prepared by reacting ethyleneand maleic anhydride in the presence of peroxide catalyst in a suitablesolvent medium. The copolymer will preferably contain substantiallyequimolar quantities of the ethylene residue and the anhydride residue.

The base EMA copolymer can be reacted with aloweralkyliminobis(loweralkylamine) which has two primary amine groupsand leads to a cross-linked EMA copolymer. The EMA copolymer should bereacted with from about 3 mole % to about 10 mole % of the cross-linkingagent. The desired pendant diloweralkylaminoloweralkylimide functionalgroups can then be incorporated into the cross-linked copolymer to alevel of at least about 3 mole % by reaction ofdiloweralkylaminoloweralkylamine with part or all of the remaining freeanhydride groups of the EMA copolymer. From about 90 mole % to about 100mole % of the diloweralkylaminoloweralkylamine preferably is used forpreparing the polyelectrolyte polymeric adsorbent employed in step (a)of the present invention whereas from about 3 mole % to about 7 mole %preferably is used for preparing the adsorbent employed in thesubsequent step (c) of the present invention. In the case of the latterpolyelectrolyte polymeric adsorbent, substantially all the remainingfree carboxyl or anhydride sites are blocked with alkoxyalkylamine asdisclosed in U.S. Pat. No. 4,157,431.

The polyelectrolyte polymeric materials used in the present inventionalso can be prepared by methods which employ the aggregation stepdisclosed in U.S. Pat. No. 4,118,554.

A preferred diloweralkylaminoloweralkylimide functional group isdimethylaminopropylimide, a preferred cross-linking agent ismethyliminobispropylamine, and a preferred alkoxyalkylamine blockingagent is methoxypropylamine.

It will be appreciated that the foregoing methods of production of thepolyelectrolyte polymeric adsorbents are for illustrative purposes onlyand that the method of fractionating blood with these materials inaccordance with the invention is not limited to any particular method oftheir preparation.

It is presently believed to be important to one aspect of the presentinvention that the exogenous heparin be employed during both adsorptionsteps with the polyelectrolyte polymers since the first adsorption stepmay also adsorb a substantial amount of the heparin from the plasmamedium when exogenously added to that medium. Generally, the amount ofheparin to add prior to the second adsorption step will be equivalent tothe amount removed by the first adsorption step. The heparin can beadded initially during the blood collection process or it can be addedto blood collected in other anti-coagulants such as ACD, CPD or CPDA.

In accordance with another aspect of the invention, the heparin can alsobe incorporated directly with the polyelectrolyte polymer beforetreatment of the blood with the polymer. In the latter case the heparinalso can be bound to the polymer by ionic bonds to form apolyelectrolyte polymer/heparin complex. The polyelectrolytepolymer/heparin complex can be conveniently formed by admixing theheparin and the polymer in aqueous suspension followed by separating theresulting complex and drying.

The starting blood plasma material can be whole blood plasma or aconcentrate thereof known to contain Factor VIII:C, for example, acryoprecipitate concentrate.

The adsorption steps of the blood fractionation process are carried outin aqueous suspension, preferably in physiological saline solution.Appropriate pH adjustments to the desired basic or acidic levels can bemade by treatment of the plasma medium with, respectively, NaOH to raisethe pH to a range of from about 7.0 to about 8.5 in adsorption step (a)of the process, and with HCl, acetic acid or preferably citric acid tolower the pH to a range of from about 5.5 to about 6.5 in adsorptionstep (c) of the process.

Separation of the adsorbed plasma fractions from the respectivesupernatants after each adsorption step can be made by filtration,centrifugation and the like separation procedures. Elution of thedesired Factor VIII:C concentrate can be had by washing the finaladsorbent with from about one to about three molar NaCl, preferably fromabout 1.5 to about 1.8 molar NaCl, and with other such physiologicallyacceptable eluants.

The Factor VIII:C concentrate can be converted to a physiologicallysuitable, sterile solid form by employing a partial desalination andconcentration (e.g., such as membrane ultrafiltration), sterilefiltration (e.g., such as by filtration through a semiporous membranehaving a pore size of about 4 microns) and subsequent lyophilization.

Factor VIII:C activity in the final recovered product can be determinedby conventional one-stage or two-stage assay methods which employmeasurement of activated partial thromboplastin time (PTT). In thesetests, the addition of partial thromboplastin to a test plasma willmeasure deficiencies of various plasma factors. The well-known one-stageprothrombin time test developed by Quick is preferred. For backgroundinformation on the one-stage assay tests see Quick, "HemorrhagicDiseases", Lea & Febiger, Philadelphia, Pa., 1957; Langdell et al., J.Lab. Clin. Med. 41, 637 (1953); and Hardisty et al.; Thromb. Diath.Haemorrh. 7, 215 (1962).

Presence of Factor VIIIR activity in the final product can be determinedby aggregation when exposed to the antibiotic substance ristocetin andmeasurement by immunoelectrophoresis or radioimmunoassay. Suitable suchassay procedures are described by Harris et al., Biochim. Biophys. Acta668, 456-470 (1981), and by Fulcher et al., Proc. Natl Acad. Sci. USA79, 1648-1652 (1982).

Although human blood is particularly described herein, it will beappreciated that other animal blood such as, e.g., bovine, porcine,equine and ovine can similarly be fractionated in accordance with thepresent invention.

The following examples will further illustrate the invention although itwill be understood that the invention is not limited to these specificexamples or the details recited therein.

EXAMPLE 1

Human donor blood was drawn into plastic blood collection bagscontaining CPDA-1 anticoagulant. Plasma was separated from the bloodserum by refrigerated centrifugation within 4 hours of collection. Fortyunits of plasma were pooled and aliquoted (200 ml) into 300 ml unittransfer bags which were then frozen and stored at -20° C.

The fresh frozen plasma was fractionated at ambient temperature in asequence of adsorption steps employing two different water-insoluble,cross-linked polyelectrolyte resins in the presence of exogenousheparin. These resins were copolymers of substantially equimolar amountsof ethylene and maleic anhydride, cross-linked with 5 mole % ofmethyliminobispropylamine, and containing pendantdimethylaminopropylimide pendant groups. The first resin, Resin A,contained 90 mole % of these pendant groups, whereas the second resin,Resin B, contained 5 mole % of said pendant groups. In Resin B all thefree carboxyl or anhydride groups were further blocked withmethoxypropylamine.

Prior to use, Resin B was preconditioned as follows:

Twelve grams of the resin were disposed in 200 ml of 0.154 M NaClcontaining 0.1% bovine serum albumin (BSA). (Human serum albumin alsocan be used instead of BSA). The pH was adjusted to 4.0 with 1.0 Mcitric acid to facilitate dispersion (about 3-5 minutes with stirring).The suspension was filtered and the filtrate discarded. The wet resincake was again dispersed in 200 ml of the NaCl/BSA solution. The pH wasadjusted while stirring to 5.8 with 1.0 M NaOH; stirring was continuedfor an additional 10 minutes. The suspension was filtered and the wetcake reserved for use in the subsequent fractionation.

All eluant and wash solutions used in the fractionation also contained0.1% BSA to minimize binding of the desired proteins to surfaces and tofurther act as a stabilizer for Factor VIII:C.

Porcine heparin, sodium salt, was employed in the fractionation bysolution in physiological saline (0.9 NaCl) to provide a solutioncontaining about 200 units/ml.

The fractionation process was carried out as follows:

One bag (i.e. 200 ml) of the fresh frozen plasma was rapidly thawed byplacing in a stirred water bath at 37° C. For the tests incorporatingheparin, one milliliter of heparin solution (i.e. 200 units) was addedto the thawed plasma in a beaker and the mixture stirred for 3-5minutes. A sample of plasma was taken for the coagulation assay. Volumeand time were noted. Control runs were made without addition of heparinto the plasma but were otherwise identical to the test runs.

Resin A (70 mg) was added to the plasma (or heparinized plasma), the pHwas adjusted to 8.0 with 1 M NaOH and maintained at this pH withstirring for 20 minutes. It was then filtered; the filtrate containedmost of the Factor VIII:C activity. The wet resin cake was resuspendedin 20 ml of distilled water, stirred for 5 minutes, filtered, and thetwo filtrates were then combined and sampled for assay of Factor VIII:Cactivity. The volume was recorded to permit calculation of totalcoagulation units present.

The pre-conditioned resin B (12 g) was then added to the filtrate (orheparinized filtrate), the pH adjusted to 5.8 with 1 M citric acid andthe suspension was stirred for 20 minutes while maintaining the pH at5.8. It was then filtered and the filter cake washed with 200 ml of0.002 M NaCl. The combined filtrates were retained for recovery ofalbumin and gamma globulin plasma fractions. The filter cake wasdispersed in 200 ml of 0.3 M NaCl, the pH adjusted to 5.8 and thesuspension was stirred for 5 minutes. The suspension was again filtered,washed on the filter with another 200 ml of 0.3 M NaCl and the filtratewas discarded.

The filter cake was then dispersed in 200 ml of eluant solution whichcontained sodium chloride (1.5 M), lysine (0.1 M) as a stabilizer, andbovine serum albumin (0.1%). The pH was adjusted to 6.0 and thesuspension was stirred for 20 minutes. The suspension was filtered, thecake optionally washed with approximately 20 ml of eluant solution, thevolume of combined filtrates noted and a sample was taken forcoagulation assay as reported in Table 1, below.

The filtrates, containing 40-70% of the original Factor VIII:Ccoagulation activity in purified form, can then be processed to achievefurther concentration and partial desalination. This can be achieved,for example, by employing a Millipore Pellicon® Cassette filtrationsystem or an Amicon DH 4 hollow fiber concentrator system, with asemi-permeable polysulfone fiber membrane having an appropriatemolecular weight cutoff for retention of the desired molecules. Theconcentrated solution can then be freeze dried and packaged for storage.These further concentration and processing steps were not, however,carried out in this specific Example 1.

The following Table 1 presents results of several fractionation runsemploying pooled CPDA-stabilized plasma and the process described above.These results demonstrate the beneficial effects of heparin addition tothe pooled plasma in combination with polyelectrolyte polymerfractionation. They illustrate a reproducible improvement in recovery ofFactor VIII:C coagulation activity both after the Resin A treatmentwhich removed Factor IX complex and in overall activity recovery (basedon the level in the original plasma) after adsorption and elution fromthe Resin B.

The Factor VIII:C determination was made by a conventional one-stage PTTassay system on an MLA Electra Coagulation Timer (Medical LaboratoryAutomation, Inc.). This instrument employs optical sensing to indicatecommencement of the clotting process. It measures the second derivativeof the coagulation rate (i.e., the rate of change of the coagulationrate). The assay was made with a reagent kit and procedure commerciallysupplied by Dade Diagnostics, Inc., which includes Factor VIII deficientplasma and ellagic acid activator as described in U.S. Pat. No.3,486,981. Coagulation times were determined for serial dilutions of thetest samples (the fractionated samples) and results were expressed aspercent of recovery of Factor VIII:C activity based on the level in theoriginal pooled plasma sample. In these runs, the pooled plasma wasassumed to contain 1 unit/ml of Factor VIII:C coagulation activity.Therefore, each of the runs was initiated with a total of 200coagulation units. The cumulative unit and percent recovery of activityafter each resin treatment is reported.

                  TABLE 1                                                         ______________________________________                                               Units                 Units   Recovery                                        Eluted                Eluted  from                                            from      Recovery from                                                                             from    Resin                                    Run No.                                                                              Resin A   Resin A (%) Resin B B (%)*                                   ______________________________________                                        CONTROL RUNS - NO HEPARIN ADDED                                               1      145       72.5        88      44.0                                     2      137       68.5        76      38.0                                     3      106       53.0        41      20.5                                     4      144       72.0        84      42.0                                     5      105       52.5        93      46.5                                     Ave    127 ± 20                                                                             63.7 ± 10.1                                                                            76.0 ± 21.0                                                                        38.2 ± 10.4                           (S.D.)                                                                        HEPARIN TREATMENT (1.03 UNITS/ML)                                             6      171       85.5        118     59.0                                     7      190       95.0        120     60.0                                     8      166       83.0        112     56.0                                     9      164       82.0        109     54.5                                     10     204       102.0       139     69.5                                     Ave    188 ± 27.0                                                                           93.9 ± 13.5                                                                            120 ± 12.0                                                                         59.8 ± 5.9                            (S.D.)                                                                        ______________________________________                                         *Based on initial plasma level                                           

EXAMPLE 2

Additional fractionation runs were made as in Example 1, above, exceptthat three different concentrations of heparin (1.0 unit/ml; 0.5unit/ml; and 0.1 unit/ml) were added to the initial plasma in step (a)prior to adsorption with Resin A and also to the effluent (filtrate) instep (c) prior to adsorption with Resin B.

The following Table 2 presents the results of these fractionation runsin terms of percent recovery of Factor VIII:C activity in which theoriginal pooled plasma was assumed to contain 1 unit/ml.

                  TABLE 2                                                         ______________________________________                                                 Amount Heparin Added                                                          1.0 unit/ml                                                                             0.5 unit/ml*                                                                            0.1 unit/ml                                      ______________________________________                                        Recovery from                                                                            91 ± 6   81        96 ± 14                                   Resin A (%)                                                                   Cumulative 53 ± 17  48        53 ± 5                                    Recovery from                                                                 Resin B (%)                                                                   ______________________________________                                         *One run only                                                            

EXAMPLE 3

In this example, polyelectrolyte polymer/heparin complexes wereinitially prepared by contacting the respective Resin A and Resin Bproducts of Example 1, above, with aqueous solutions of sodium heparinand drying the resulting complexes. These resin/heparin complexes werethen used to fractionate blood according to the procedure of Example 1without further exogenous addition of heparin solution.

Initially, porcine heparin, sodium salt (1003 units dissolved in 50 mlof water) was added to each of:

(A) 300 mg of Resin A suspended in 100 ml of water, and

(B) 50 g of Resin B suspended in 200 ml of water.

In each case, stirring was maintained at ordinary room temperature (ca.22°-25° C.) for about one hour. The resin/heparin complexes wereisolated by filtration on Whatman #54 cellulosic filter paper (98%retention efficiency at 20-25 microns according to the manufacturer)followed by washing three times with 100 ml portions of water to removeany unbound heparin. The resin/heparin complexes were then dried priorto employment in the blood fractionation process.

Plasma was fractionated substantially as described in Example 1 exceptthat the resin/heparin complexes were used in place of the correspondingresins and further exogenous addition of heparin was omitted.

In this example, recovery of Factor VIII:C coagulation activity afterthe Resin A/heparin complex treatment was quantitative within the limitsof ordinary experimental error (117%). The overall recovery afteradsorption and elution from the Resin B/heparin complex was 78.4% of thelevel in the original plasma in this particular example.

In the foregoing Examples 1-3, Resin A was made substantially accordingto methods with reactants and molar proportions as described in Example1 of U.S. Pat. Nos. 4,097,473 and 4,118,554 and Example 12 of U.S. Pat.No. 4,157,431. Resin B was made substantially according to methods withreactants and molar proportions as described in Example 1 of U.S. Pat.No. 4,157,431.

EXAMPLE 4

Substantially similar results as obtained in Examples 1-3, above, areobtained when diethylaminoethylamine is substituted for an equivalentamount of dimethylaminopropylamine, and/or when ethyliminobisethylamineis substituted for an equivalent amount of methyliminobispropylamine,and/or when ethoxyethylamine is substituted for an equivalent amount ofmethoxypropylamine in said Examples 1-3.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention and it is intended that all such otherexamples be included within the scope of the appended claims.

What is claimed is:
 1. A method for the preparation of a concentrate ofFactor VIII:C from blood plasma which comprises(a) admixing said bloodplasma or a concentrate thereof at pH of from about 7.0 to about 8.5with from about 0.01% to about 0.1% by weight of water-insoluble,polyelectrolyte copolymer of ethylene and maleic anhydride cross-linkedwith from about 3 mole % to about 10 mole % ofloweralkyliminobis(loweralkylamine) and containing from about 90 mole %to about 100 mole % of pendant diloweralkylaminoloweralkylimidefunctional groups, said admixing in the presence of exogenous heparin,(b) separating the supernatant from the resulting adsorbed plasmafraction, (c) admixing said supernatant at pH of from about 5.5 to about6.5 with from about 1% to about 10% by weight of water-insoluble,polyelectrolyte copolymer of ethylene and maleic anhydride cross-linkedwith from about 3 mole % to about 10 mole % ofloweralkyliminobis(loweralkylamine), containing from about 3 mole % toabout 7 mole % of pendant diloweralkylaminoloweralkylimide functionalgroups, and further characterized in that substantially all theremaining free carboxyl or anhydride sites are blocked withalkoxyalkylamine, said admixing in the presence of exogenous heparin,(d) separating the resulting adsorbed plasma fraction from thesupernatant and recovering therefrom a concentrate of Factor VIII:C byelution from the adsorbent, and (e) wherein said alkyl and alkoxy havefrom about one to about four carbon atoms and the exogenous heparin ispresent in a range of from about 0.01 to about two units per ml ofplasma.
 2. The method of claim 1 in which the copolymer of ethylene andmaleic anhydride is cross-linked with methyliminobispropylamine and inwhich the pendant diloweralkylaminoloweralkylimide functional group isdimethylaminopropylimide.
 3. The method of claim 1 in which the heparinlevel in steps (a) and (c) is from about 0.1 to about one unit per ml.4. The method of claim 1 in which the concentration of thepolyelectrolyte copolymer in step (a) is about 0.03-0.04% and in step(c) is about 5-6%.
 5. The method of claim 1 in which the copolymer ofethylene and maleic anhydride is cross-linked withmethyliminobispropylamine, the pendant diloweralkylaminoloweralkylimidefunctional group is dimethylaminopropylimide, the heparin level in steps(a) and (c) is from about 0.1 to about one unit per ml and theconcentration of polyelectrolyte copolymer in step (a) is about0.03-0.04% and in step (c) is about 5-6%.
 6. The method of claim 1 inwhich the exogenous heparin is incorporated in solution with the bloodplasma in step (a) and the supernate in step (c).
 7. The method of claim5 in which the exogenous heparin is incorporated in solution with theblood plasma in step (a) and the supernate is step (c).
 8. The method ofclaim 1 in which the exogenous heparin is precomplexed with thepolyelectrolyte copolymers used in steps (a) and (c).
 9. The method ofclaim 5 in which the exogenous heparin is precomplexed with thepolyelectrolyte copolymers used in steps (a) and (c).